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Trends in Recombinant Proteins Manufacturing
and strength of uorescence depend on the molecular oxygen concentration present around the dye
in the environment. The emitted uorescence is collected and transmitted for interpretation outside of the bioreactor. These electrodes work better than the traditional platinum probe electrodes
to detect molecular oxygen, and they can be used in both liquid and gas phases. PreSens (www.
pres ens.de) is an example of a noninvasive oxygen sensor that measures the partial pressure of
DO and gaseous oxygen. Sensor spots are xed on the inner surface of glassware or transparent
plastic material (disposables). Therefore, molecular oxygen concentration can be measured in a
noninvasive and nondestructive manner from outside through the vessel wall. Different coatings for
different concentration ranges are available. It offers online monitoring of DO concentration, ranging from 1 ppb to 45 ppm, with dependence on ow velocity and oxygen measurement in the gas
phase. These coatings can be autoclaved.
Ocean Optics (www.ocea nopt ics.com) offers the world’s rst miniature spectrometer with a wide
array of sensors for oxygen and pH detection in the gas phase.
pH sensors act based on their absorption or uorescence characteristics. For ber- optic pH
measurements are carried out based on both uorescence- and absorbance- based pH indicators. The
most common dyes for uorescence- based measurements are 8- hydroxy- 1,3,6- pyrene trisulfonic
acid and uorescein derivatives, while phenol red and cresol red are used for absorption- type
measurements. Fluorescent dyes are sensitive to ionic strength, which limits their use for broadrange pH measurement (for pH beyond 3 U).
CO2 sensors work on the principle of pH measurement for a carbonate buffer embedded in a CO2permeable membrane. The reaction time of the sensor is long, and quaternary ammonium hydroxide
provides a faster response.
Fluorescence- based sensors are attractive because they facilitate the development of portable
and low- cost systems that can be easily deployed outside of the laboratory environment. Such
measurements are insensitive to changes in dye concentration, leaching, and photobleaching of the
uorophore and instrument uctuations, unlike unreferenced uorescence intensity measurements.
The performance of the sensor system is characterized by a high degree of repeatability, reversibility, and stability.
4.3.5.2 Biomass Sensors
Information about the biomass concentration can be obtained using turbidity sensors. Generally,
these sensors are based on the principle of scattered light. Most turbidity sensors have the disadvantage of a linear correlation only for low particle concentrations, but sensors that use backscattering
light (180°) have linear properties for high particle concentrations. A translucent window is necessary in disposable bioreactors in order to check for the desired wavelength in the IR region. The S3
Mini- Remote Futura line of biomass detectors (www.appl ikon bio.com) incorporates sensors inside
disposable bioreactors. This sensor system uses an ultra- lightweight pre- amplier for connecting to
the ABER disposable probe (www.bio proc ess- eng.co.uk/ prod uct/ aber- fut ura- pico/ ).
4.3.5.3 Electrochemical Sensors
Electrochemical sensors include potentiometric, conductometric, and voltammetry sensors. Thick-
and thin- lm sensors and chemically sensitive eld- effect transistors (ChemFETs) possess the
potential as potentiometric disposable sensors in bioprocess control because they can be produced
inexpensively and in large quantities.
Many pH- sensing systems rely on amperometry methods, but they require constant calibration
owing to instability or drift. The setups of most amperometry sensors are based on the pH- dependent
selectivity of membranes or lms on the electrode surface.
While turbidity sensors detect total biomass concentration, capacitance sensors provide information specically about viable cell mass. Electrical capacitance and conductance generally characterize the electrical properties of cells in an alternating electrical eld. Cell membrane integrity exerts
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a signicant inuence on the electrical impedance to estimate only viable cells. The Biodis Series
by Hamilton (www.hami lton comp any.com) and Aber (www.aber inst rume nts.com) for monitoring
viable biomass in disposables applications is available. An integrated version is manufactured from
Eppendorf (www.eppend orf.com).
4.3.5.4 Pressure Sensors
Pressure is another important process parameter frequently monitored during bioprocess unit
operations such as ltration, chromatography, and many other procedures. A traditional stainlesssteel pressure gauge can be used in conjunction with a single- use experimental setup, but this
combined setup has the drawback that the pressure gauge must be sterilized separately. Furthermore,
the connection of the sensor to the previously gamma- irradiated single- use assembly could raise
problems.
Many bioprocess unit operations have in- built pressure- control systems to avoid signicant
pressure- related safety issues. In traditional stainless- steel reactors, pressure is monitored and
tightly controlled, as pressure can inuence mass transfer and prevent contamination. Moreover,
a high- pressure event is a potentially hazardous situation. A clogged vent lter in a bioreactor can
easily cause rupture of the bags, spillage of the reactor’s contents, and exposure of the operators to
unprocessed bulk.
Another application where pressure monitoring is central to process performance is depth and
sterile ltration. A lter’s ability is primarily measured by either ow decay or pressure increase.
However, adding reusable traditional pressure transducers to a process train fails the purpose of
using a single- use process setup. Depending on the process application, the contact surface of a traditional device’s product requires either sanitization or moist heat sterilization.
Traditional devices are sterilized through SIP, where the product contact surface is exposed to
steam sterilization in devices that can be placed in an autoclave, and the entire device is exposed
to the steam. However, many single- use process components are not compatible with moist heat
sterilization temperatures, which necessitates separate sterilization of the stainless- steel device and
possibly less than that for an optimal connection to a pre- sterilized single- use assembly.
Single- use pressure sensing facilitates rapid changeover of product contact surfaces in both
development applications and early- phase clinical manufacturing. For example, single- use pressure
sensors from PendoTECH (www.pendot ech.com) were designed to enable pressure measurement
with single- use assemblies that have exible tubing as the uid path. These single- use pressure
sensors are gamma- irradiation compatible (up to 50 KGy), and the uid path materials meet
United States Pharmacopoeia (USP) Class VI guidelines and are compliant with EMEA 410 Rev 2
guidelines.
The USP Class VI designation is considered the most stringent and, therefore, most useful for
medical applications. It involves the following three evaluations for in vivo biological reactivity,
generally performed on mice or rabbits to mimic use in humans:
• Acute systemic toxicity (systemic injection) test: This test measures toxicity and irritation
when a sample of the compound is administered orally, applied to the skin, or inhaled.
• Intracutaneous test: This test measures toxicity and localized irritation when the sample is in
contact with live subdermal tissue (specically, the tissue intended to be in contact with the
medical device).
• Implantation test: This test measures toxicity, infection, and irritation of intramuscular
implantation of the compound into a test animal model over several days.
The compound will be assessed in these three tests in order to conrm that it has an extremely low
toxicity level, and it will be subjected to several temperature assessments for set periods. Materials
that meet USP Class VI standards generally have a high- level quality and better compliance with
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the US FDA because such materials will carry a substantially lower risk of causing harm to patients
from reaction to a toxic material.
USP Class VI Testing is only one standard of biocompatibility. However, although not a limited
series of tests, some biocompatibility requirements for medical devices may exceed the testing
performed in USP Class VI. ISO- 10993 is a more rigorous standard for the biological evaluation of
medical devices.
ISO- 10993 is a standard that involves systemic toxicity and intracutaneous reactivity testing.
However, it also tests for additional cytotoxicity, genotoxicity, chronic toxicity, hemocompatibility,
and, more importantly, systemic toxicity. A different level of ISO- 10993 testing is primarily
required for medical devices that will be permanently or semi- permanently implanted into a patient.
Therefore, for devices that are not intended to be implanted or will have limited contact with patients,
ISO- 10993 testing may be more extensive than necessary.
In a single- use bioreactor, a sensor can be installed on a vent line to measure headspace pressure.
Even though the sensors are qualied to be used up to 75 psi, the core sensor shows accurate values
in the low- pressure range required for a single- use bioreactor.
4.3.5.5 Sampling Systems
Continuous sampling from a bioreactor can be accomplished using a sterile lter and a peristaltic
pump to obtain a cell- free sample. A presterilized sampling container, which contains a needleless
syringe that can be welded to the bag bioreactor’s sampling module, is available for use. A sample
is pumped into the container of these assemblies. The sampling containers can be removed when
needed, and the tube is heat- sealed. Other sampling systems have a presterilized Leuer connection,
including a one- way valve, which prevents the sample from owing back into the reactor. The
sample is withdrawn from the reactor using a syringe and directed to a reservoir through a sample
line. For example, Cellexus Biosystems (https:// celle xus.com) and Millipore (www.sigma aldr ich.
com) use such sampling systems. The Cellexus system connected to the sample line can have
up to six sealed sample pouches. The reservoir sample can then be pushed into the pouches,
which are subsequently separated by a mechanical sealer, which results in sealed, sterile samples.
Several sampling manifolds with a customizable option are offered by bioreactor manufacturers/
suppliers.
The proprietary Millipore system comprises a port insert that can be tted to several bioreactor
side ports and several exible conduits that can be opened and closed individually for sampling.
These ports are connected to exible, single- use sampling containers. Sampling is limited to the
number of available conduits in each module.
These sampling systems allow aseptic sampling but are limited in terms of the number of samples
collected per module and the lack of automation. While these methods help obtain good validation
data, the risk of contamination is not completely removed because the bioreactor is breached every
time a sample is withdrawn. Therefore, there is a need to develop or choose other methods that will
not require contact with the media.
4.3.5.6 Connectors
The complexity of bioprocessing makes it difcult to design systems without any weaknesses;
contamination is associated with a risk that requires all connectors, tubes, and implements to join
various steps of a process and perform sampling in a sterile environment. Single- use components
were rst applied in connectors and lines, as cleaning was a challenging task. Unlike hard piping,
the exible tubing used in single- use transfer lines does not require costly and time- consuming
cleaning and validation procedures. This allows manufacturers to quickly alter the process steps
or convert the resultant over to a new product. This feature is a key advantage for multiple production facilities wherein process requirements change depending on the type of drug being produced.
Innovative manufacturers now incorporate single- use tubing assemblies throughout the bioprocess,
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starting from seed trains to the nal ll applications. Additional cost savings result from the need for
reduced labor and the chemical, water, and energy demands associated with cleaning and validation.
Yet, in hard- walled systems, SIP systems are used only because steam is used for CIP/ SIP
operations. Even then, the risk of contamination persists. As much of the SUT systems in these
applications are being used in the biomedical eld, the device industry had always been ahead of the
regulatory requirements. Biocompatibility issues have long been resolved, and vendors can provide
detailed information on their devices that might be needed by regulatory agencies. As manufacturing
of these devices is a complex process, it is unlikely for a user to request custom design devices;
however, the diverse choices available today can adequately modify any system that would use an
off- the- shelf item. As before, emphasis is being placed on the importance of an off- the- shelf item
over custom designs. Tube connectors and sealers are newer entrants as single- use bags for mixing
and bioreactors have become more popular; yet, there is a limited choice of suppliers, mainly Cytiva
LifeSciences Sartorius Stedim Biotech. The cost of this equipment is still high, but then the alternative is to use expensive aseptic connectors. Generally, if a good choice of aseptic connectors is
available, then such connectors should be preferred over tube connectors, as heat- activated systems
always create issues related to poor connection. Additionally, the use of aseptic connectors allows
connecting tubes that may not be thermolabile.
Modern bioprocessing facilities scale up inoculum from a few million cells in several milliliters
of culture to production volumes of thousands of liters. This process requires an aseptic transfer at
each point along the seed train. Traditional bioprocessing facilities accomplish the scale- up process
using a dedicated series of stainless- steel bioreactors linked together with valves and rigid tubing. To
prevent contamination between production runs, a CIP system is designed in each bioreactor, vessel,
and piping line to remove any residual materials. Such CIP and SIP systems require extensive validation testing, and the valves and piping present in these systems can create additional validationrelated challenges.
Advances in SUT systems have allowed bioprocess engineers to replace most storage vessels and
xed piping networks with single- use storage systems and tubing assemblies, respectively. Singleuse systems eliminate the need for CIP validation for many components and reduce maintenance and
capital costs by eliminating the need for expensive vessels, valves, and sanitary piping assemblies.
Single- use media storage systems are routinely used for volumes ranging from 20 L to 2,500
L. Media storage systems are generally sterilized by gamma irradiation by their manufacturers
(before installation at the bioprocess facility) and are often tted with integrated lters, sampling systems, and connectors. The use of single- use digital- to- analog connectors (DACs) or tube
welders and sealers with compatible tubing allows operators to make sterile connections between
the presterilized SUBs for aseptic transfer of media, cells, and any other liquid required to be added.
The DACs can also be used for downstream applications. These aseptic connectors can be used for
high ow and high- pressure applications.
Similarly, customized presterilized single- use tubing assemblies are used to transfer inoculum
between bioreactors using a peristaltic pump or by applying headspace pressure. Flexible tubing
with aseptic connectors is used as transfer lines between the bioreactors in the process. Such transfer
lines reduce the number of reusable valves required for transfer and eliminate problem areas for
CIP and SIP validation. Terminating each presterilized transfer line with a single- use SIP connector
provides sterility assurance equal to the sterility in traditional xed piping at lower capital costs.
In some instances, liquids are transferred from a higher to a lower ISO environment, and strong
assurance for sterility (there should not be cross- contamination during the transfer) is needed; therefore, a conduit can be installed in the walls connecting the two areas, with the cleaner room having
a higher pressure. A pre- sterilized tube is then inserted from the side of the lower ISO class to side
of the higher ISO class, thereby forming a connection between the vessels, and the liquid is then
transferred through a peristaltic pump. Upon completion of the transfer, the tube is pulled into the
higher ISO class area and nally discarded. This method helps establish a connection between the
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downstream and upstream areas without the risk of contamination during transfer to a lower ISO
class area, such as a downstream area.
4.3.5.7 Tubing
Flexible tubes are an essential part of all single- use systems and are subject to safety concerns
described in an earlier chapter on leachables and extractables. Several attributes of exible tubing
require evaluation, namely heat resistance, operating temperature range, chemical resistance, color,
density, shore hardness, exibility, elasticity, surface smoothness, mechanical stability, abrasion
resistance, gas permeability, sensitivity to visible and UV light, composition of layers, weldability,
sealability, and sterilizability by gamma irradiation or in an autoclave.
All tubes used in bioprocessing conform to USP Class VI classication, FDA 21 CFR
177.2600, and EP 3A Sanitary Standard. In cGMP manufacturing, these are classied as bulk
pharmaceuticals.
4.3.5.8 Pumps
Pumps are used for uid transfer by generating hydrostatic pressure or differential pressure; the
maximum allowable working pressure would be determined at the weakest part of the bioprocess
component exposed to the pressure. In some unit operations such as harvesting, TFF, and chromatography, the molecule is highly sensitive to any changes in the pumping process. Pulsing of
pressure can affect the uid being pumped or even damage the pump parts. The pump must meet the
following criteria for suitability with the intended use:
• Low volume and minimal surface area exposure.
• Low levels of leachables and extractables.
• Controlled ow and pressure.
• Low shear and pulsation.
• No mechanical spalling/ shedding of contact materials.
• Self- priming.
• No heat buildup.
• Sterility.
• High volumetric efciency.
Permanent stainless- steel process lines are not only expensive to install and are complex but
also require extensive cleaning and validation. Some of the pumps use mechanical seals that cannot
maintain constant ow or sterility, which makes them less suitable for handling biologics.
Currently, single- use pumping solutions include peristaltic pumps, syringe pumps, and diaphragm pumps. Single- use positive displacement quaternary diaphragm pumps are one of the best
options for bioprocessing applications. These are volume displacement pumps, easy to use, and
avoid contact with the product; however, they can exert stress on the tubing, especially when they are
being operated for a prolonged period. The stress on the tube may lead to erosion of particles from
the tube and contaminate the uids being passed through. Many biological drugs are shear- sensitive,
and peristaltic pumps can help preserve these drugs by applying low pressure and providing gentle
handling. By contrast, a piston pump’s valve system stimulates fast ow through small orices,
which potentially causes damage to the biological products. Even valveless piston pumps apply high
pressures and high shear factors, ultimately harming a biological product.
High- end peristaltic dispensing pumps are advantageous in terms of an improved pulsation- free
pump head design, precise drive motor, and state- of- the- art calibration algorithm. They are exceptionally accurate at microliter ll volumes. Peristaltic pumps with single- use tubing eliminate crosscontamination and do not require cleaning validation because the tubing is the only part that comes
into contact with the product. Likewise, cleaning validation of peristaltic pumps with single- use
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tubing is signicantly easier than that of piston pumps. On the contrary, viscous products can be
a problematic issue for peristaltic pumps. Peristaltic pumps apply only approximately 1.3 bar of
pressure, and their accuracy diminishes when they handle products with viscosity higher than 100
cP. Several improvements have been made to pumps intended for downstream processing, including
HPLC, TFF, and virus ltration applications, which enable high process yields throughout the
pressure range (e.g., quantum; www.wat son- mar low.com/ us- en/ range/ wat son- mar low/ sin gle- usepumps/ quan tum/ ). Single- use pumps usually consist of bags instead of stainless- steel vessels and
use special agitators, single- use tubing, coupling aids, and valves. Single- use components reduce
the cost of cleaning and eliminate extensive validation. Plug- and- play options are available for TFF
applications. These pumps provide a linear ow across the pressure range required for the process;
they induce ultra- low shear, thereby increasing the downstream process yield.
A diaphragm pump is a positive displacement pump that uses a combination of the reciprocating
action of a rubber, thermoplastic, or Teon diaphragm and suitable nonreturn check valves to pump
a uid. Quaternary diaphragm pumps are driven one after another by connector plates that move
back and forth. These pumps are ideal for handling all liquid biologics, including viscous liquids.
Some pumps have the ability to self- prime, run dry, be operated at a constant ow, involve only low
shear and pulsation, and not involve any heat accumulation.
4.3.5.9 Tube Welder and Sealers
In scenarios where it is possible to use a thermoplastic tube, welding offers an easy, inexpensive,
and very secure solution. Examples of thermoplastic tubes include C- Flex, PharMed, and Bioprene.
Thermoplastic tubes must be aseptic, have the same dimensions (inner diameter and outer diameter), and have their ends capped. The thermoplastic tubes are placed in opposite directions, parallel
to each other, and they can be simultaneously sealed by cutting across the tubes using a heated
blade. The blade should be preheated to achieve the welding temperature, achieve sterility, and
dehydrogenize the blade before the welding process. The dehydrogenization procedure normally
lasts for 30 s at 250°C or for 3 s at 320°C. After the tubes are being cut across, they are moved
against each other so that the ends of each tube connected to the aseptic systems are positioned
directly opposite to each other on either side of the blade. A duration of a welding cycle can range
from 1 to 4 min, depending on the material and tube diameter. The main welding systems available
today include Sterile Tube Fuser (GE Healthcare), BioWelder (Sartorius Stedim), Aseptic Sterile
Welder 3960 (SEBRA, www.sebra.com), TSCD (Terumo, www.terumo tran sfus ion.com), and SCD
11B (Terumo— Terumo supplies its equipment mainly for blood transfusion purposes). Both GE
Healthcare and Sartorius Stedim lead the installations in the bioprocessing industry.
When disconnecting an aseptic connection, the ends of the connection must be capped with
aseptic caps, and this should be performed inside a laminar hood or by using tube sealers; some
of the examples include products from PDC (www.pdc biz.com), Saint- Gobain (www.saint- gob ain.
com), Sartorius Stedim (www.sartor ius- ste dim.com), Cytiva (www.cyt ival ifes cien ces.com/ en/ us),
Terumo (www.terumo tran sfus ion.com), and SEBRA (www.sebra.com). Most of these sealers can
seal tubes with a diameter ranging from 0.25 inch to approximately 1.5 inch, and the sealing process
can take 1– 4 min. Most of the sealers operate on an electrical heating element, but radio- frequencies
are also used for sealing tubes. There is no need to use a laminar ow hood for these procedures. In
most instances, applying a crimper in two places and cutting the tube between the crimps offers the
cheapest solution.
4.3.6 saMPling
During manufacturing, sampling is routinely performed to assure compliance by validating inprocess parameters such as pH, DO, OD, pCO2, and so on. Most of the single- use systems have
one or more integrated sampling lines, partly equipped with special sampling valves, sampling
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manifolds, or special sampling systems. A popular single- use sampling valve is the Clave connector
from ICU Medical (www.icu med.com), which is also used in intravascular catheters for medical
applications. Through the sampling valve, it is easy to collect a sample using a Luer- Lok syringe.
The dynamic seal present inside the valve guarantees that the sample can be collected only when the
syringe is connected, thereby ensuring that the sample comes into contact with only the valve’s inner
aseptic parts. However, the samples drawn do not remain sterile.
Manifolds consisting of sampling bags, sampling asks, or syringes are appropriate for collecting
aseptic samples in single- use systems. These manifolds can be connected to the systems through
aseptic connectors or tube welding. Sampling manifolds allow multiple sampling over a given
period for quality purposes. The main feature of the manifold is that the number of manipulations
in a process can be signicantly reduced. The manifold systems are delivered ready for process
use in a preassembled and sterile manner. Only one connection is sufcient to allow several bags
to be lled.
Additionally, sampling can also be carried out using manifold systems, where sample containers
of a manifold are arranged in parallel, and the last container is used as a waste container. The initial ow and the subsequent sample are guided to their respective containers using Y- , T- , or X- hose
barbs and tube clamps. SIP connections, as expected, also allow the connection of manifold systems
to conventional stainless- steel processing equipment.
4.3.7 doWnstReaM PRocessing
SUT is an attractive solution for minimizing downtimes between batches, additional burden on
cleaning, validation of these procedures, and, most importantly, risk of contamination between
batches. SUTs also facilitate easy switching between product lines in a multiproduct facility. SUTs
such as columns, certain disposable hardware systems, and single- use ow paths have been successfully used for upstream processing and have become an integral part of evolution with downstream
processing.
Single- use liquid chromatography systems, such as ÄKTA ready XL chromatography systems,
which have disposable ow paths and prepacked columns, can support large- scale commercial manufacturing and conveniently meet the capacity starting from single- use 2000 L upstream
processes with a high titer. These systems are very useful in both technology transfer and process
scale- up operations.
An increased emphasis is being placed on supporting therapeutic drugs to be more affordable.
Single- use systems and CM operations are critical drivers for the decrease in the overall manufacturing and investment cost to make this a possibility.
The adoption of single- use components in downstream bioprocessing has been an evolutionary process with a few revolutionary peaks occasionally. Initially, buffer bags and devices
were being used for normal ow ltration, including ltration of virus and guard lters in chromatographic columns. Yet, gradually, more complex concepts were introduced, including singleuse devices for TFF and chromatography during downstream processing. Today, the industry
has arrived at the consensus that, while many of the upstream operations can be converted to
fully single- use systems, at least some elements of downstream processing will still be carried
out in the traditional manner, and the reasons cited for this assertion are as follows: (1) columns
and resins will always be too expensive to throw away, and (2) because columns can have a
very large size, nding a suitable single- use substitution will be highly challenging. However,
as pointed out by historical evidence, the same arguments were presented only 15 years ago,
opposing bioreactors’ conversion to single- use devices. Today, downstream processing science
is developing more rapidly than upstream science; more recently, the use of membrane adsorbs
has been recommended for large- scale purication of antibodies. These membranes are much
cheaper than classical resins.
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4.3.7.1 Cell Harvest
65
For cell harvesting and debris removal, ltration is an alternative approach to conventional centrifugation. Currently available single- use ltration systems offer exibility and scalability of operations.
They are advantageous in terms of the ease of scale- up and the availability of presterilized lter
capsules that can be integrated directly into production lines. Although this stage is generally carried
out by centrifugation or lenticular ltration, depth lter systems (e.g., Millipore Pod lters) have
provided the rst available alternative in single- use lenticular lters; these adsorptive depth lters
combine two distinct separation technologies into one efcient operation to enhance ltration
ability and retention while compressing multiple ltration steps. Depth lters use a porous ltration
medium to retain particles throughout the medium, rather than just on the surface of the medium.
Depth lters are made of bers in the form of a mesh that is spread out on a substrate; special
additives such as activated carbon, ceramic bers, and other such specic components are embedded
with a binder to form the lter. Depth lters use their entire depth to retain the particles based on
sieving compounded by adsorption effects, unlike retentive lters where the ltered material is
concentrated on the surface. These lters are commonly used when the uid to be ltered has a
high load of particles because, compared with other types of lters, they can retain a large mass of
particles before clogging.
Scale- up is achieved by inserting multiple pods into a holder, with formats allowing 1– 5 or
5– 30 pods as required. Further single- use depth lter formats include the Stax- System from Pall
Life Sciences, encapsulated Zetaplus from Cuno, and L- Drum from Sartorius Stedim, Millipore
Clarisolve, double– open- end high capacity, and extended open- end high- capacity adsorptive depth
lters for primary and secondary clarication. These lters allow efcient cell clarication by reducing the cell biomass, host cell protein (HCP), and host DNA and removing most of the cell debris
to enable easy loading in the chromatographic column.
The performance of depth lters depends on the colloid content of the bioreactor ofoad and the
cell debris removal ability of the upstream centrifuge. Usually, depth lters are operated at a constant ow of 100– 200 L/ (m2 h) and up to 150 L feed/ m2 of lter depending on the composition of the
feed stream. The Millipore Millistak+ Pod depth lter has a maximum lter area of 33 m2, resulting
in a batch capacity of 3– 5,000 L. The Millipore Mobius FlexReady process equipment supports a
larger lter area (55 m2). As washing of these lters requires very large volumes of buffers, holding
tanks of appropriate size can be lined with single- use PE liners.
In some instances, crossow ltration of high volumes of claried harvest is performed to reduce
the volume for subsequent purication; however, debris buildup extends the total time taken for the
ltration process. While this process is not sterile, the use of a single- use lter prevents the problem
of cross- contamination.
Single- use continuous centrifugation devices such as Ksep® are available for processing recombinant proteins and vaccines. The Ksep is a closed continuous- ow centrifuge that works by creating centrifugal force and the feed- ow force. This system offers the benet of efcient processing
without affecting recovery because of its low shear, continuous operation.
Each technology has its advantages and drawbacks; therefore, testing each option, and choosing
the appropriate one based on the specic method and cell type is recommended. The single- solution
performance depends on the USP performance, cell density, viability, and the extent of the cell
debris present in the bioreactor broth.
4.3.7.2 Purification
For protein isolation and purication, a steel column is packed with a resin (stationary phase) comprising porous beads made of a polysaccharide, mineral, or synthetic matrix conjugated to specic functional groups exploiting different separative principles. The protein mixed with other
components is loaded onto the column slowly. Once the protein is bound to the resin, the resin is
eluted with solutions of appropriate pH and containing required electrolytes to separate the target
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protein from the mixture. The resin is cleaned and sanitized for repeated use, and this process may
involve dozens or, perhaps, hundreds of cycles.
Several vendors now offer columns (e.g., ReadyToProcess™ columns by GE Healthcare) for
use in ÄKTA machines to overcome the time needed to pack the resin and operate a column.
GE Healthcare offers a wide range of resins and custom resins. These are high- performance
bioprocessing columns that are prepacked, prequalied, and presanitized. The ReadyToProcess™
chromatographic columns and the use of single- use or single- use ow paths eliminate the risk of
cross- contamination. The ÄKTA ready system has a sanitary design and is well suited for use in
a cGMP- regulated environment. The simple procedures and low downtime between products and
batches of ÄKTA readily facilitate improved economy and productivity. Other prepacked columns
such as the ReadyToProcess™ columns include Opus (Repligen), GoPure (Life Technologies).
The ÄKTA system is designed for seamless scalability, delivering the same performance level as
that achieved with conventional processing columns such as AxiChrom™ and BPG™. The ÄKTA
system is currently available with a range of BioProcess™ media in four different sizes (1, 2.5, 10,
and 20 L), and these columns are designed to purify biopharmaceuticals for clinical phase I and II
studies. Depending on the scale of operations, they can also be used for full- scale manufacturing
and preclinical studies. The columns can be used in a wide range of chromatographic applications
to separate various compounds such as proteins, endotoxins, DNA, plasmids, vaccines, and viruses.
Single- use chromatography solutions such as ÄKTA XL systems are available as prepacked
columns, single- use ow paths, plug- and- play chromatography columns, and membranes, as well
as presterilized lters and tubing to eliminate cleaning validation. ÄKTA ready chromatography
systems are designed for process scale- up and manufacturing, and they operate through ready- touse, single- use ow paths, thereby eliminating cleaning validation between products and batches.
Purication of proteins from complex mixtures is a key process in pharmaceutical research and
production. However, protein purication using chromatography based on particulate matrices is a
lengthy procedure and takes longer separation times. Several ligands are available (Table 4.2).
Membrane adsorbers are advantageous in removing high- molecular- weight contaminants such as
DNA and viruses during monoclonal antibody manufacturing. Such contaminant molecules do not
readily diffuse into traditional resins; thus, most of the purication steps relying on column chromatography require dramatically oversized columns. The hydrodynamic benets of oversized columns
provide the opportunity to operate membrane adsorbers at much greater ow rates than those for
columns, thereby considerably reducing buffer consumption and shortening the overall process
time by up to 100- fold. Commercially used membrane adsorbers are Mustang® (Pall), Sartobind®
TABLE 4.2
Different Types of Membranes and Ligands
Membrane Type Description Ligand
Sulfonic acid (S) Strong acidic cation exchanger R- CH2- SO3- >3
Quaternary ammonium (Q) Strong basic anion exchanger R- CH2- N+ (CH3)
Carboxylic acid(C) Weak acidic cation exchanger R- COO- >3
Diethylamine (D) Weak basic anion exchanger R- CH₂- N(C2H5)
Phenyl Hydrophobic interaction (HIC) Phenyl >3
IDA Metal chelate Iminodiacetic acid >3
Protein A Afnity Protein A 0.45
Epoxy- activated Coupling Epoxy group 0.45
Aldehyde- activated Coupling Aldehyde group 0.45
Source: Sartorius Stedim
Pore Size
(µm)
3
2
>3
>3
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Trends in Recombinant Proteins Manufacturing
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(Sartorius), ChromaSorb® (Millipore), and Adsept® (Natrix). These membranes are commonly used
for removing process- related impurities such as DNA and endotoxin in the ow- through mode.
The accelerated seamless antibody purication process is an entirely single- use continuous
downstream process for mAb production, based on ÄKTA periodic counter- current chromatography
(PCC) Protein- A, mixed- mode, and anion exchange resin columns. In this process, all three columns
are cycled simultaneously. These systems offer the advantage of both single- use and continuous processing in a single application while providing exibility, ease of operation, and increased capacity.
When selecting single- use consumables, it is crucial to ensure that the supply chain is strong.
Ensuring the right documentation and testing for extractables and leachables aligns with following
regulatory compliance.
Single- use systems provide great exibility to handle several products in a facility; the fast turnaround time between batches or products results in a quicker product release.
4.3.7.3 Virus Removal
Virus contamination is a risk to all biotechnology products derived from cell lines of human or
animal origin. Contamination of a protein product with endogenous viruses from cell banks or
adventitious viruses from personnel can have profound clinical implications. Three complementary
approaches assure viral safety in licensed biological products:
• Thorough testing of the cell line and all raw materials for the presence of viral contaminants,
• Assessment of the ability of downstream processing to clear infectious viruses, and
• Testing of the product at appropriate steps for the presence of contaminating viruses.
A combination of methods based on inactivation, adsorption, and size exclusion are available. The FDA requires demonstration of virus clearance by two methods. Examples of inactivation procedures are the use of solvents and detergents, chemical treatments, low pH, or microwave
heating. Adsorption- based methods include chromatography, and virus removal by mechanical or
molecular size exclusion is executed by normal (forward) and TFF methods.
Ion exchange and protein A chromatography methods are widely used to remove viruses, and
several key studies have been conducted in collaboration with the FDA. Yet, the developer is responsible for proving the suitability of any method for virus removal. Membrane ltration has been used
for viral clearance in mAb production processes for many years. Hollow ber membrane cartridges
and even surface- modied, hydrophilic membranes with high void volume and minimal fouling
capable of reducing high viral titers are some of the recent single- use options for viral clearance.
Adsorptive lters can be used at the end of the purication process in line with the viral ltration
step. These lters combine the principles of size exclusion and adsorption to retain aggregates by
hydrophobic interactions while increasing viral ltration efciency. Several manufacturers such as
Sartorius, Pall, and Millipore offer single- use virus ltration solutions to remove large enveloped
viruses and small nonenveloped viruses. Nano- sized lters are commonly used as viral removal
lters. The most common virus retention of these lters is of the size 20 or 50 nm.
4.4 FILTRATION: ULTRAFILTRATION/ DIAFILTRATION AND TANGENTIAL
FLOW FILTRATION
Filtration applications are well suitable for single- use processing. Ultraltration and dialtration are
used to concentrate and change the buffer of a solution. During the nal formulation, ultraltration
and dialtration are used to transfer the active pharmaceutical ingredient to a stabilizing environment and achieve the correct concentration of the product. A volume of up to 300– 5,000 L may need
to be processed, depending on whether the column eluates can be fractionated. Membranes with a
30- kDa molecular weight cutoff are often used to retain antibodies, and the process intermediate is
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